Design and synthesis of dual acting carbamate inhibitors of acetylcholinesterase and monoamine oxidase

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Design and synthesis of dual acting carbamate

inhibitors of acetylcholinesterase and

monoamine oxidase

M Lourens

22094873

Dissertationsubmitted in fulfillment of the requirements for the

degree

Magister Scientiae

in

Pharmaceutical Chemistry

at the

Potchefstroom Campus of the North-West University

Supervisor:

Dr ACU Lourens

Co-supervisor:

Prof A Petzer

Assistant Supervisor:

Prof JP Petzer

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Financial assistance towards this study, contributed by the National Research Foundation (NRF) is hereby acknowledged. Opinions, findings, conclusions and recommendations are those of the author and are not necessarily to be contributed to the NRF.

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ACKNOWLEDGEMENTS

 All the glory is to God.

 My supervisor, Dr. A.C.U. Lourens, I am greatly thankful for your support, guidance and patience.

 My co-supervisors, Prof. J.P. Petzer and Prof A. Petzer for all your advice and inputs.  My parents, Freddie and Annette Lourens, for your unconditional love and support.

Thank you for allowing me to pursue my dreams.

 My husband, Reinald Landro, for believing in me and supporting me all the way. Thank you for walking this journey with me.

Jeremiah 29:11

‘For I know the plans I have for you, declares the Lord, plans to prosper

you and not to harm you, plans to give you hope and a future.’

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i

4.4.4 Method………. 110 4.4.5 Results………. 111 4.4.6 Discussion of results……….. 112 4.5 Summary……… 113 CHAPTER 5: CONCLUSION………..……… 114 BIBLIOGRAPHY………... 118 ADDENDUM………..……… 153 1 H NMR AND13C NMR SPECTRA……….……… 154 MASS SPECTRA………..…...……… 203

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v INFRA-RED SPECTRA………...……… 215 HPLC TRACES……..………..………… 229

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vi

ABSTRACT

TITLE

Design and synthesis of dual acting carbamate inhibitors of acetylcholinesterase and monoamine oxidase.

KEY WORDS

Carbamate, Dihydroquinolinone, Acetylcholinesterase, Monoamine Oxidase, Alzheimer’s disease, Parkinson’s disease, Parkinson’s disease dementia.

BACKGROUND AND RATIONALE

Alzheimer’s disease (AD), the most common neurodegenerative disorder, affects about 10% of the population over the age of 65 years. The disease is typified by symptoms such as memory loss and impairment in abilities including attention, concentration, orientation and judgment. Two hypotheses exist regarding the pathogenesis of AD: the first proposes that a decrease in the production of acetylcholine (Ach) in the synaptic junction (cholinergic hypothesis) is the main causative factor, while the second suggests that the disease is largely due to the aggregation of toxic amyloid-β (Aβ) peptide in the brain (amyloid hypothesis).

Ach is a neurotransmitter which plays an important role in attention, cognitive processing and other cognitive functions. Consequently, cholinesterases are very important enzymes as they modulate the levels of Ach in the brain. Acetylcholinesterase (AChE) inhibitors including donepezil, rivastigmine and galantamine, inhibits the metabolism of Ach, thus increasing the Ach available for binding. These agents are therefore most commonly used in the symptomatic treatment of AD. Currently, no AD agent is registered as neuroprotective, and this neurodegenerative disorder progresses unhindered with an associated decrease in quality of life. Therefore, the discovery of novel drugs that can slow or stop its progression is of great importance.

Parkinson’s disease (PD), on the other hand, is the second most common neurodegenerative disorder and is characterised by the loss of dopamine (DA) in the nigrostriatum. The symptoms of PD can by classified as either motor or non-motor symptoms. Motor symptoms include bradykinesia, muscle rigidity, resting tremor and impaired postural balance. Non-motor symptoms include sleep disturbances, autonomic

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vii dysfunction and sensory abnormalities. Parkinson’s disease dementia (PDD), which is typified by cognitive decline, is often experienced during the later stages of the disease. With the increase in life expectancy throughout the Western world, it is expected that PDD will become more prevalent in the future.

The symptomatic treatment of PD usually involves levodopa use. Its continuous use is unfortunately associated with motor complications that impair the quality of life. Additional therapies, which include catechol-O-methyltransferase inhibitors, DA agonists and monoamine oxidase (MAO) B inhibitors are further used in PD treatment. The MAO enzymes (MAO-A and MAO-B), play an important role during the oxidative degradation of amine neurotransmitters including DA, serotonin (5-HT) and epinephrine. The inhibition of these enzymes decreases DA metabolism in the brain, resulting in an increase in the DA concentration. It is also likely to be neuroprotective as the production of harmful metabolic by-products such as hydrogen peroxide is decreased. Since the use of non-selective, irreversible MAO inhibitors are associated with severe side-effects, current efforts are aimed at designing selective, reversible MAO inhibitors. The MAO-B enzyme is of particular significance in PD because it is more active than MAO-A in the basal ganglia which is mainly responsible for the catabolism of DA in the brain. The treatment of the cognitive deficits experienced during the later stages of PD, and in PDD, in particular is not adequate and treatments that address the motor and non-motor aspects simultaneously are urgently required.

It is postulated that a dual MAO-B and AChE inhibitor would improve motor symptoms of PD while improving cognitive deficits at the same time. Such an agent would not only be useful in the treatment of PD, but also in PDD and AD as the MAO inhibition component has the possibility of offering neuroprotection. The dihydroquinolinone scaffold has been shown to be privileged with regards to the inhibition of MAO, while the carbamate moiety often features in the structures of AChE inhibitors.

AIM

The aim of this study was therefore to design, synthesise and evaluate novel carbamates as dual inhibitors of MAO and AChE.

METHODS

Compounds were synthesised using a one step literature procedure. 6-Hydroxy-3,4-dihydro-2(1H)-quinolinone, 7-hydroxy-3,4-dihydro-6-Hydroxy-3,4-dihydro-2(1H)-quinolinone, 3- and 4-acetamidophenol were reacted with commercially available carbamoyl chlorides under basic conditions to yield

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viii the target compounds. Compounds were characterised by using NMR and IR spectroscopy as well as mass spectrometry. Purity was determined by HPLC and melting points were determined. The potential of the synthesised compounds to inhibit MAO enzymes were expressed as IC50 (50% inhibition concentration) values and the SI (selectivity index) was determined. A fluorometric assay, using kynuramine as substrate was employed. AChE inhibitory activity was determined by measuring the rate of thiocholine production, as generated by the hydrolysis of acetylthiocholine, which served as substrate in a spectrophotometric assay.

RESULTS AND DISCUSSION

Twenty eight novel compounds were successfully synthesised, albeit in low yields. Generally, most of the synthesised compounds exhibited weak to no inhibition of both MAO-A and MAO-B. Compound 8g, 2-oxo-1,2,3,4-tetrahydroquinolin-7-yl methyl(phenyl)carbamate, was the most potent MAO-B inhibitor of the current series with an IC50 value of 3.73 µM, and was MAO-B selective. It is postulated that the rigidity of the carbamate side chain is responsible for the loss of activity observed for the compounds of this study, when compared to the highly potent dihydroquinolinone derivatives of a previous study. Disappointingly, none of the synthesised compounds inhibited AChE, possibly due to the replacement of an ionisable amine group with an amide. Although the biological results of this study were disappointing, useful information was obtained regarding structural requirements for binding to both MAO and AChE.

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ix

OPSOMMING

TITEL

Ontwerp en sintese van dubbelwerkende karbamaat inhibeerders van asetielcholienesterase en monoamienoksidase.

SLEUTELWOORDE

Karbamaat, Dihidrokinolinoon, Asetielcholienesterase, Monoamienoksidase, Alzheimer se siekte, Parkinson se siekte, Parkinson se siekte demensie.

AGTERGROND EN MOTIVERING

Alzheimer se siekte, die mees algemene neurodegeneratiewe siekte, affekteer ongeveer 10% van die bevolking bo die ouderdom van 65 jaar. Die siekte word gekenmerk deur simptome soos geheueverlies en inkorting van vermoëns soos aandag, konsentrasie, oriëntasie en oordeel. Twee hipoteses bestaan aangaande die patogenese van Alzheimers se siekte: volgens die eerste is 'n afname in die produksie van asetielcholien in die sinaptiese spleet (cholinerge hipotese) die belangrikste veroorsakende faktor, terwyl die tweede hipotese voorstel dat die siekte grootliks te wyte is aan die aggregasie van giftige amiloïed-β peptied (Aβ) in die brein (amiloïede hipotese).

Asetielcholien is 'n neurotransmitter wat 'n belangrike rol in aandag, kognitiewe prosessering en ander kognitiewe funksies speel. Gevolglik is cholienesterases baie belangrike ensieme aangesien hulle die vlakke van asetielcholien in die brein moduleer. Asetielcholienesterase (AChE)-inhibeerders wat donepesil, rivastigmien en galantamien insluit, inhibeer die metabolisme van asetielcholien, en verhoog sodoende die asetielcholien wat beskikbaar is vir binding. Hierdie is dus die mees gebruikte middels in die simptomatiese behandeling van Alzheimers se siekte. Tans is daar geen anti-Alzheimer’s middel wat geregistreer is as neurobeskermend nie, en die verloop van hierdie neurodegeneratiewe siekte gaan dus ongehinderd voort met 'n gepaardgaande afname in lewenskwaliteit. Die ontdekking van nuwe middels wat die siekte kan vertraag of stop, is dus van groot belang.

In teenstelling daarmee is Parkinson se siekte die tweede mees algemene neurodegeneratiewe siekte en word dit gekenmerk deur ’n verlies aan dopamien in die nigrostriatum. Die simptome van Parkinson se siekte kan geklassifiseer word as óf motories of nie-motoriese simptome. Motoriese simptome sluit bradikinesie, spierstyfheid, rustremore en verswakte posturale balans in. Nie-motoriese simptome sluit weer slaapversteurings,

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x outonome disfunksie asook sensoriese abnormaliteite in. Parkinson-se-siekte-demensie, wat gekenmerk word deur kognitiewe agteruitgang, word dikwels ervaar gedurende die latere stadiums van die siekte. Met die toename in lewens verwagting regdeur die Westerse wêreld, word daar verwag dat Parkinson-se-siekte-demensie meer algemeen in die toekoms sal wees.

Die simptomatiese behandeling van Parkinson se siekte behels gewoonlik die gebruik van levodopa. Ongelukkig lei die langdurige gebruik daarvan tot motoriese komplikasies wat lewensgehalte benadeel. Addisionele terapie, wat die gebruik van katesjol-O-metieltransferase-inhibeerders, dopamienagoniste en MAO B inhibeerders insluit, word verder gebruik in die behandeling van die siekte. Die MAO ensieme (MAO-A en MAO-B), speel 'n belangrike rol in die oksidatiewe afbraak van amienneurotransmitters soos dopamien, serotonien en epinefrien. Die inhibisie van hierdie ensieme verminder dopamienmetabolisme in die brein, wat gevolglik lei tot 'n toename in die dopamienkonsentrasie. Dit is waarskynlik ook neurobeskermend aangesien die produksie van skadelike metaboliese byprodukte soos waterstofperoksied verminder word. Aangesien die gebruik van nie-selektiewe, onomkeerbare MAO-inhibeerders geassosieer word met ernstige newe-effekte, is huidige navorsingspogings gemik op die ontwerp van selektiewe, omkeerbare MAO-inhibeerders. Die MAO-B ensiem is van besondere belang in Parkinson se siekte, aangesien dit meer aktief is as MAO-A in die basale ganglia wat hoofsaaklik verantwoordelik is vir die katabolisme van dopamien in die brein. Die behandeling van die kognitiewe defekte wat voorkom gedurende die later stadiums van Parkinson se siekte en in Parkinson-se-siekte-demensie, is veral onvoldoende en behandeling wat die motoriese en nie-motoriese aspekte gelyktydig kan aanspreek is uiters nodig.

Daar word gepostuleer dat 'n tweeledige MAO-B- en AChE-inhibeerder die motoriese simptome van Parkinsons se siekte sal behandel, terwyl verbetering van kognitiewe tekorte terselfdetyd sal plaasvind. So 'n middel sal dus nie net gebruik kan word in die behandeling van Parkinson se siekte nie, maar ook in Parkinson-se-siekte-demensie en Alzheimer se siekte, aangesien die MAO-inhibisie komponent die bykomende voordeel van neurobeskerming kan bied. In vorige studies is aangetoon dat die dihidrokinolonoonkern geassosieer word met die inhibisie van MAO, terwyl die karbamaatgroep dikwels voorkom in die strukture van AChE-inhibeerders.

DOEL

Die doel van hierdie studie was dus om karbamate te ontwerp, te sintetiseer en te evalueer as tweeledige inhibeerders van MAO en AChE.

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xi METODES

Verbindings is gesintetiseer deur gebruik te maak van 'n eenstap literatuurmetode. Die teikenverbindings is verkry deur 6-hidroksi-3,4-dihidro-2(1H)-kinolinoon, 7-hidroksi-3,4-dihidro-2(1H)-kinolinoon, 3- en 4-asetamidofenol met kommersieel beskikbare karbamoїelchloriede onder basiese toestande te laat reageer. Verbindings is gekarakteriseer deur gebruik te maak van KMR en IR-spektroskopie asook massaspektrometrie. Suiwerheid is bepaal deur HPLC en smeltpunte is bepaal. Die vermoë van die gesintetiseerde verbindings om die MAO ensieme te inhibeer is uitgedruk as IC50 (50% inhibisie konsentrasie) waardes terwyl die SE (selektiwiteitsindeks) ook bepaal is. ’n Fluorometriese toets, met kinuramien as substraat, is gebruik. AChE inhiberende aktiwiteit is bepaal deur die tempo van tiocholien produksie te bepaal, wat ontstaan as gevolg van die hidrolise van die asetieltiocholiensubstraat, in 'n spektrofotometriese toets.

RESULTATE EN BESPREKING

Agt-en-twintig verbindings is suksesvol gesintetiseer, alhoewel opbrengste oor die algemeen laag was. In die algemeen het die meeste van die gesintetiseerde verbindings swak of geen inhibisie van beide MAO-A en MAO-B getoon. Verbinding 8g, 2-okso-1,2,3,4-tetrahidrokinolien-7-iel metiel(feniel)karbamaat, was die mees potente MAO-B inhibeerder van die huidige reeks met 'n IC50 waarde van 3.73 μM, en die verbinding is MAO-B selektief. Daar word gepostuleer dat die rigiditeit van die karbamaatsyketting verantwoordelik is vir die verlies aan aktiwiteit wat waargeneem is vir die verbindings van hierdie studie, in vergelyking met die hoogs potente dihidrokinolinone wat gesintetiseer is in ‘n vorige studie. Nie een van die gesintetiseerde verbindings het ongelukkig AChE geïnhibeer nie, moontlik as gevolg van die vervanging van 'n ioniseerbare amiengroep met 'n amied. Hoewel die biologiese resultate van hierdie studie teleurstellend was, is nuttige inligting verkry met betrekking tot strukturele vereistes vir binding aan beide MAO en AChE.

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xii

LIST OF ABBREVIATIONS

5-HT Serotonin Aβ Amyloid-β/Aβ Ach Acetylcholine AChE Acetylcholinesterase

AChEI Acetylcholinesterase inhibitor AD Alzheimer’s disease

ADH Aldehyde dehydrogenase

Amplex Red N-Acetyl-3,7-dihydroxyphenoxazine APCI Atmospheric-pressure chemical ionisation APOE Apolipoprotien E

APOEԑ2 Apolipoprotein ԑ2 APOEԑ3 Apolipoprotein ԑ3 APOEԑ4 Apolipoprotein ԑ4

APP Amyloidprecursor protein

BBB Blood-brain barrier BuChE Butyrylcholinesterase

ChAT Choline acetyltransferase ChEI Cholinesterase inhibitor COMT Catechol-O-methyltransferase

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xiii D1 DA type 1 receptor D2 DA type 2 receptor D3 DA type 3 receptor D4 DA type 4 receptor D5 DA type 5 receptor DA Dopamine

DEPT Distortionless enhancement by polarization transfer DLB Dementia with Lewy bodies

DMF Dimethylformamide

DMSO Deuterated dimethylsulfoxide DTNB 5,5’-Dithiobis-(2-nitrobenzoic acid)

EtOAc Ethyl acetate

FAD Flavin adenine dinucleotide FADH2 Reduced FAD

Fe2+ Ferrous ion

GSH Glutathione

H2O2 Hydrogen peroxide

HMBC Heteronuclear multiple bond correlation HPLC High pressure liquid chromatography

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xiv HRMS High resolution mass spectra

HSQC Heteronuclear single quantum correlation

IC50 Half maximal inhibitory concentration IGF Insulin-like growth factor

IR Infrared

J Coupling constant

KCl Potassium chloride

LRRK2 Leucine-rich-repeatkinase-2

MAO Monoamine oxidase

MAO-A Monoamine oxidase isoform A MAO-B Monoamine oxidase isoform B mAChR Muscarinic acetylcholine receptor MAPT Microtubule-associated protein tau MCI Mild cognitive impairment

ml millilitre

mM Millimolar

mmol Milimole

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xv N Equivalence per liter

NA Noradrenaline

nAChR Nicotinic acetylcholine receptor

NaH Sodium hydride

NaOH Sodium hydroxide NFT Neurofibrillary tangles NH4+ Ammonia

nM Nanomolar

NMDA N-methyl-D-aspartate

NMR Nuclear magnetic resonance NO Nitric oxide

NPS Neuropsychiatric symptoms NSP Neuritic senile plaques

O2 Oxygen

O2•- Radical superoxide OH• Hydroxyl radical

OXPHOS Oxidative phosphorylation

PD Parkinson’s disease

PDD Parkinson’s disease dementia

Pl Phosphoinositol

PIGD Postural instability gait difficulty PINK1 PTEN-induced purative kinase 1

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xvi PSEN-1 Presenilin 1

PSEN-2 Presenilin 2

ROS Reactive oxygen species

SD Standard deviation SET Single electron transfer SI Selectivity index

SNpc Substantia nigra pars compacta SORL1 Sortilin-related receptor gene

TLC Thin layer chromatography Tyr Tyrosyl residues

δ Chemical shift

λex Excitation wavelength λem Emission wavelength

μl Microliter

μM Micromolar

Kinetics

E Enzyme

[E] Enzyme concentration ES Enzyme-substrate complex

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xvii [I] Inhibitor concentration

[S] Substrate concentration

Kd Equilibrium dissociation constant Ki Inhibition constant

Km Michaelis-Menten constant vi Initial reaction velocity Vmax Maximum velocity

NMR

δ Delta scale used to indicate chemical shift J Coupling constant br d Broad doublet br s Broad singlet br t Broad triplet d Doublet dd Doublet of doublets

ddd Doublet of doublets of doublets

m Multiplet

p Pentet

ppm Parts per million

q Quartet

s Singlet

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xviii

LIST OF FIGURES

Figure 2.1 The synthesis and metabolism of DA……….. 33

Figure 2.2 Oxidative deamination of monoamines by mitochondrial MAO………… 44

Figure 2.3 Single electron transfer (SET) mechanism as proposed by……… 45

Figure 2.4 Polar nucleophilic mechanism as proposed by……….. 46

Figure 2.5 The chemical structures of some MAO-A inhibitors……….. 48

Figure 3.1 The chemical structures of a dihydroquinolinone (30), ladostigil (3) and paracetamol (31)………. . 52 Figure 3.2 Experimental setup for the synthesis of dihydroquinolinone-carbamates (8c, f, g; 9c, d) and acetamidophenol-carbamate derivatives (10a-f, h; 11a-g, i)……… . . . . 56 Figure 3.3 Experimental setup for the synthesis of dihydroquinolinone-carbamates (8a-b, d-e; 9a-b) and acetamidophenol-carbamate derivatives (10g; 11h)………... . . . . 57 Figure 4.1 Transformation of a substrate through an enzyme catalytic reaction…… 89

Figure 4.2 The Michaelis-Menten plot showing the effect of substrate concentration on Vi……….. . 90 Figure 4.3 The Lineweaver-Burk plot………. 91

Figure 4.4 Lineweaver-Burk plot of competitive inhibition………... 92

Figure 4.5 Lineweaver-Burk plot of non-competitive inhibition………... 93

Figure 4.6 The oxidation of kynuramine to 4-hydroxyquinoline……….. 95

Figure 4.7 A summary for the method followed to determine IC50 values………..… 98 Figure 4.8 AChE activity determination through the reaction of thiocholine and

DTNB……….………...

.

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xix Figure 4.9 Flowdiagram representing the method for determination of IC50 values

for the inhibition of AChE………...

.

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xx

LIST OF TABLES

Table 1.1 Structures of compounds selected for synthesis………... 6 Table 2.1 Summary of Parkinson’s disease dementia………... 37 Table 2.2 Summary of MAO inhibitors……….. 47 Table 3.1 Chemical structures of dihydroquinolinone-carbamates (8a – g, 9a – d)

and acetamidophenol-carbamate derivatives (10a – h, 11a - i) that were successfully synthesised in this study……….

. . ..

58 Table 3.2 NMR data and HMBC correlations of

2-oxo-1,2,3,4-tetrahydroquinolin-7-yl piperidine-1-carbox2-oxo-1,2,3,4-tetrahydroquinolin-7-ylate (8a)………

.

64 Table 3.3 NMR data and HMBC correlations of

2-oxo-1,2,3,4-tetrahydroquinolin-6-yl diphen2-oxo-1,2,3,4-tetrahydroquinolin-6-ylcarbamate (9c)………...

.

66

Table 3.4 NMR data and HMBC correlations of

4-(acetylamino)phenyldimethylcarbamate (10b)………...

.

68 Table 3.5 NMR data and HMBC correlations of 3-(acetylamino)phenyl

dipropan-2-ylcarbamate (11h)………..

.

69 Table 3.6 The experimentally determined and calculated high resolution masses

of the various synthesised quinolinone-carbamatederivates (8a-g, 9a-d)………

. . .

70 Table 3.7 The experimentally determined and calculated high resolution masses

of the various synthesised acetomidophenol-carbamatederivates (10a-h, 11a-i)………

. . .

71 Table 3.8 HPLC analysis results of the quinolinone-carbamatederivates (8a-g,

9a-d)………

.

72 Table 3.9 HPLC analysis results of the acetomidophenol-carbamatederivates

(10a-h, 11a-i)………..

.

73 Table 4.1 IC50 values of the synthesised compounds for the inhibition of human

MAO-A and MAO-B………

.

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xxi Table 4.2 Comparison of IC50 values of the compounds synthesised in this study

with those of the 3,4-dihydro-2(1H)-quinolinones reported in literature….

.

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xxii

LIST OF SCHEMES

Scheme 2.1 The Fenton reaction….……… 21

Scheme 2.2 The metabolism of ACh to choline and acetic acid as catalysed by cholinesterases (ChE)……….

.

23 Scheme 2.3 Selegiline and its metabolites………. 49 Scheme 2.4 Rasagiline and its metabolite………. 50 Scheme 3.1 The general synthetic route for quinolinone-carbamates and

acetamidophenol derivatives……….. 55 Scheme 3.2 The general synthetic route for dihydroquinolinone-carbamates and

acetamidophenol derivatives……….. 56

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1

CHAPTER 1 INTRODUCTION

1.1 INTRODUCTION AND OVERVIEW

Dementia is defined as a progressive deterioration in cognition, function and behaviour. The symptoms of dementia can be ascribed to damaged brain cells or damaged brain cell connections. Without these cells functioning correctly, one’s memory, behaviour and ability to reason are therefore changed (Alzheimer’s-Association, 2016). The most common neurodegenerative disorder, namely AD, accounts for approximately 70% of all cases of dementia (Reitz & Mayeux, 2014) and affects about 6% of the population over the age of 65 (Guzior et al., 2015). PD on the other hand, is classically viewed as a motor-disease, but it is estimated that 40% of PD patients are likely to develop dementia (Emre, 2003). Alongside the worldwide increase in life expectancy, the prevalence of dementia (and age related neurodegenerative disorders), is expected to rise, resulting in an escalating economic and social burden (Dorsey et al., 2007; Muller & Woitalla, 2010; Winter et al., 2010; Tom et al., 2015).

AD is classified as a progressive neurodegenerative disorder characterisedby memory loss and behavioural changes (Carter et al., 2012), the presence of neurofibrillary tangles, senile plaques, cholinergic neuron loss and neuronal atrophy (Balin & Hudson, 2014; Karch & Goate, 2015). There are two hypotheses regarding the pathogenesis of AD. Firstly, it has been suggested that the onset of AD is due to the decrease of ACh production in the synaptic junction (cholinergic hypothesis) (Fisher, 2012), and secondly, that the aggregation of toxic Aβ peptide in the brain results in the development of AD (amyloid hypothesis) (Selkoe, 2001). The cholinergic hypothesis proposes that treatments that prevent cholinergic deficits through inhibition of acetylcholine esterase (AChE) will result in improved cognition (Fisher, 2012).

Cholinergic neurotransmission is affected mainly by the neurotransmitter ACh, which binds to muscarinic and nicotinic receptors (Fisher, 2012). The neurotransmitter is metabolised by both AChE and butyrylcholinesterase (BuChE) (which also hydrolyses other choline esters), resulting in choline and acetate, thus preventing further activation of ACh receptors (Stoddard et al., 2014). Acetylcholinesterase inhibitors (AChEIs), including donepezil,

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2 rivastigmine and galantamine are thus most commonly used in the symptomatic treatment of AD (Burke, 2015).

In addition to decreased cholinergic neurotransmission and the development of senile plaques, the presence of oxidative stress contributes to brain tissue damage and progression of AD (Di Carlo et al., 2012). MAO is an enzyme responsible for the catabolism of monoamines, such as DA, adrenaline and noradrenaline (NA) (Youdim et al., 1988). During the metabolic reaction catalysed by the two isoforms (MAO-A and MAO-B) of this enzyme, hydrogen peroxide (H2O2) and subsequently, oxidative free radicals, are produced, which increases oxidative stress. The MAO-B levels in AD patients are up to 3-fold higher in the frontal, temporal and parietal cortex compared to the MAO-B levels in controls (Saura et

al., 1994, Nebbioson et al., 2012, Huang et al., 2015). Inhibition of MAO-B will thus

potentially result in decreased levels of reactive oxygen species (ROS) and neurotoxic products. The therapeutic potential of MAO-inhibitors in AD treatment is thus due to their potential neuroprotective properties and goes beyond their effect on neurotransmission (Guzior et al., 2015).

Conversely, PD is a neurodegenerative disorder characterised by the loss of DA in the nigrostriatum. The main manifestations associated with PD are motor symptoms, including akinesia or bradykinesia, rigidity and tremor being directly related to dopaminergic striatal loss (Hornykiewicz, 2008). In this case, MAO-B inhibitors, such as rasagiline and selegiline, are used in the symptomatic treatment of PD. Inhibition of MAO-B results in a decrease in the degradation of DA, thereby increasing synaptic DA (Lees, 2005) and improving motor symptoms (Knoll, 2000). As in AD, in addition to their role in the symptomatic treatment of PD, MAO-B inhibitors may also afford neuroprotection (Guzior et al., 2015).

PDD is a complication of PD that arises in the late stages of the disease and is diagnosed by the identification of deficits in recognition memory, attention processes and visual perception as well as visual hallucinations and cognitive fluctuations (Emre et al., 2007). According to Aarsland et al. (2001a), 100 out of every 100 000 patients suffering from PD develops PDD. At the time of PD diagnosis, the risk and rate of cognitive decline are associated with the patient’s age, for which the risk of cognitive impairment and developing dementia are much greater (Aarsland & Kurz, 2010).

The determination of the pathological mechanisms that contributes to PDD is complicated due to the heterogeneity of the disease and involves limbic and neocortical Lewy body deposition, with neurofibrillary tangles and senile plaques playing a role in some patients. Dysfunction of non-dopaminergic neurotransmitter systemsalso occurs (Williams-Gray et al., 2007). In PDD, as in AD, there is a substantial reduction in cortical cholinergic activity. There

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3 is no cure for PDD, and current symptomatic treatment, which include the use of cholinesterase inhibitors (ChEIs), provides only modest relief (Ballard et al., 2011).

In both AD and PD (particularly in PDD), the combined inhibition of both MAO and AChE will thus be beneficial (Bautista-Aguilera et al., 2014). Inhibition of AChE will result in enhanced cognition, due to an increase in cholinergic neurotransmission (Stoddard et al., 2014), while MAO inhibition will potentially afford neuroprotection and also improve motor symptoms in PD (Guzior et al., 2015).

1.2 RATIONALE

The dihydroquinolinone scaffold has been established as privileged for the inhibition of MAO-B, e.g (1, 2) (Meiring et al., 2013).

1 IC50 MAO-A: 53.7 µM 2 IC50 MAO-A: 22.5 µM

IC50 MAO-B: 0.191 µM IC50 MAO-B: 2.33 µM

The carbamate moiety on the other hand, occurs in the structures of potent inhibitors of AChE e.g. ladostigil (3) and rivastigmine (4) (Li et al., 2014).

3 Ladostigil 4 Rivastigmine

IC50 AChE: 0.011 µM IC50 BuChE: 1.28 µM

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4 Of particular interest to this study, was work done by Roy and co-workers (Roy et al., 2012) who synthesised a novel series of tetrahydroquinolinecarbamates with promising AChE inhibitory activities (e.g. 5, 6).

5 IC50 AChE: 3.31 µM

6 IC50 AChE: 2.57µM

A limited number of dual targeted AChE-MAO-B inhibitors have also been designed and synthesised previously, e.g. N-methyl-N-((1-methyl-5-(3-(1-(2-methylbenzyl)piperidin-4-yl)propoxy)1H-indol-2-yl)methyl)prop-2-yn-1-amine (7) with anti-cholinesterase and MAO-A and -B inhibitory activities (Bautista-Aguilera et al., 2014), providing proof of concept. This approach is particularly advantageous because it addresses the multifactorial nature of neurodegenerative diseases such as AD and PD by targeting both the MAO and AChE enzymes.

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5 7 IC50 AChE: 2.8 µM

IC50 BuChE: 4.9 µM IC50 MAO-A: 6.3 µM IC50 MAO-B: 183.6 µM

As previously stated, the prevalence of AD and PD, which are common, incurable neurodegenerative diseases (Alves et al., 2008) are increasing, indicating the relevance of research in this regard. Although many research efforts have been aimed at relieving the plight of AD sufferers, the only therapies existing are symptomatic (Fereshtehnejad et al., 2014). Similarly, only inadequate symptomatic treatment is available for the treatment of PD or PDD (Ballard et al., 2011). AD is a multifactorial disease (Huang & Mucke, 2012), while PD is likewise characterised by heterogeneous neuropathology (Kehagia et al., 2010). Therefore, compounds that interact with multiple targets, such as AChE and MAO-B, could be particularly effective as they would be complementary.

1.3 HYPOTHESIS OF THIS STUDY

It is postulated that the combination of the dihydroquinolinone and carbamate moieties will result in chemical entities with potent dual MAO-B and AChE inhibitory activities. It is further hypothesised that the acetamidophenol derivatives, as indicated in table 1, will also exhibit dual activity since similar functionalities are present in these molecules. These compounds will find potential application in the treatment of AD and PD while also possibly providing neuroprotection.

1.4 AIM OF THIS STUDY

The main aim of this study will be to combine the carbamate and dihydroquinolinone moieties in one molecule in order to obtain a polyfunctional entity with inhibitory activity against both MAO and AChE.

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6 Compounds will be synthesised in one step using literature procedures. The structures of proposed derivatives are indicated in table 1.

Table 1.1 Structures of compounds selected for synthesis

C7 substituted dihydroquinolinone derivatives

8a 8b

8c _8d

8e 8f

8g

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7 9a 9b 9c 9d 4-acetamidophenol derivatives 10a 10b 10c 10d

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8 10e 10f 10g 10h 3-Acetamidophenol derivatives 11a 11b 11c 11d

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9

11e 11f

11g _11h

11i

1.5 OBJECTIVES OF THIS STUDY The objectives of this study are to:

Synthesise dihydroquinolinone carbamates in an attempt to obtain potent dual MAO/AChE inhibitors. Since acetamidophenol is structurally related to dihydroquinolinone, some acetamidophenol derivatives will also be synthesised in order to assess the necessity of the closed dihydroquinolinone ring system. The selection of carbamoyl chlorides will be based on commercial availability.

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10 Evaluate the synthesised compounds in vitro as inhibitors of AChE and MAO. Kynuramine and 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) will be used as substrates for MAO and AChE, respectively.

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11

CHAPTER 2 LITERATURE REVIEW

2.1 ALZHEIMER’S DISEASE INTRODUCTION

AD was first described by Alois Alzheimer who was a psychiatrist at the Frankfort Psychiatric Hospital. From 1901 until 1906, one of his patients presented with symptoms such as disturbance of memory, aggression, paranoia, crying, sleep disorder and progressive confusion. The symptoms showed fast progression with increased intensity. After the death of the patient he reported “A peculiar severe disease process of the cerebral cortex”, now known as Alzheimer’s disease (Hippius & Neundorfer, 2003; Ferri et al., 2006; Balin & Hudson, 2014).

AD can be described as a multifactorial (dependant on a number of factors, in particular genetic or environmental factors) (St George-Hyslop & Petit, 2005) and heterogeneous (diverse in character) (Selkoe, 2001) disorder characterised by a decline in cognitive function and a progressive loss of memory (McKhann et al., 1984). AD affects approximately 6 - 10% of people aged 65 or older in the United States (Geldmacher, 2007) and the risk of developing AD is related to age, with a 8% chance of developing the disease for people over the age of 85 (Reitz, 2014). It is the most common neurodegenerative disease, and accounts for more than 70% of all cases involving neurodegeneration (Reitz & Mayeux, 2014).

AD occurs in two forms: an early onset form (genetically determined (Balin & Hudson, 2014), appearing before the age of 65) accounting for 1-5% of AD cases (Reitz & Mayeux, 2014) and a late onset form (more common (Balin & Hudson, 2014) and causes dementia amongst the elderly over the age of 65) responsible for more than 95% of all AD cases (Reitz & Mayeux, 2014). AD can be symptomatically characterised by memory loss and behavioural changes (Balin & Hudson, 2014) and neuropathologically by the presence of neurofibrillary tangles, senile plaques, cholinergic neuron loss (that is irreversible) and neuronal atrophy in specific brain regions (Zhu et al., 2007; Balin & Hudson, 2014; Reitz & Mayeux, 2014). AD can be classified as mild, moderate or severe and the classification determines the type of treatment. In mild AD, patients have difficulty with cognition and cannot recall short-term memory and new learnings, but can function alone with no caregiver (Forstl & Kurz, 1999; Geldmacher, 2007). Patients with mild AD have however an increased risk for developing

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12 depression. Patients with moderate AD struggle with activities of daily living and require close supervision with noticeable loss in memory. These patients can also experience wandering, insomnia and delusions. In severe AD, a patient has a noticeable loss of activities of daily living with limited language abilities, agitation, incontinence and increased dependence on a caregiver with almost all cognitive function impaired (Geldmacher, 2007). The average duration of AD is between 4 and 8 years, but the illness can last up to 20 years (Mayeux, 2003; Reitz, 2014). According to the Alzheimer’s association (2016), it is essential to determine the precise biological changes that occur during AD and why the progression differs in patients in order to prevent, slow or even stop AD (Alzheimer's-Association, 2016). 2.1.1 Incidence and diagnosis of Alzheimer’s disease

The incidence of AD seems to be increasing as the life expectancy of the population increases (Hurd et al., 2013). The worldwide estimation of AD affected individuals is 17 million (Reitz, 2014). The onset of AD can occur as early as the age of 40 years; however AD mainly manifests in patients over the age of 60. In early stages, it is often difficult to distinguish between AD symptoms and normal aging. It is also quite common for the misdiagnosis of AD in its later stages due to the possibility of various different mental illnesses (Khachaturian, 1985).

Sperling et al. (2011) suggest that there is an opportunity for disease modification if therapeutic intervention is applied during early onset of AD. However, there is no direct test that can be used for the determination or diagnosis of AD. Instead, various approaches, which include taking a family and medical history, asking family members or other persons of interest if there are any notable behaviour or skill changes and blood tests, to rule out any other potential causes of dementia, are used (Alzheimer's-Association, 2016). Laboratory tests that can be helpful for AD diagnosis in pre-mortem patients, in conjunction with PET, MRI or other imaging methods, include the study of the levels and the presence of Aβ 1-40/1-42 peptides and the total number of modified tau protein in cerebrospinal fluid (Hampel

et al., 2004; Risacher et al., 2009; Prvulovic & Hampel, 2011; Teipel et al., 2013; Balin &

Hudson, 2014). The diagnosis is also based on the results obtained from general physical and neurological examinations as well as a cognitive test (Alzheimer's-Association, 2016). 2.1.2 Symptoms of Alzheimer’s disease

AD symptoms progress gradually from mildly impaired memory functions to severe cognitive loss (Ferris & Farlow, 2013). The disease initially develops after the age of approximately 60 and worsens with an increase in age (Reitz, 2014). Mild AD symptoms include:

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13 forgetfulness, short-term memory loss, a repetitive asking of questions, a loss of interest in hobbies, impaired instrumental functions and anomia, where moderate AD presents with a progression in cognitive deficits, aphasia, dysexecutive syndrome, impairment in basic activities of daily living, which results in a transition into care. Finally, in severe AD, symptoms such as agitation and an altered sleep pattern is present, resulting in a total dependence on care for normal daily activities such as dressing, feeding and bathing (Ferris & Farlow, 2013). According to the Alzheimer’s Association (2016), in the severe state, patients may not recognise their own family members, losing their ability to communicate, leaving them bed-bound and completely dependent on full-time care. These patients are also more vulnerable to infections which can be life threatening (Alzheimer's-Association, 2016).

The symptoms of AD differ between patients, with deterioration in the ability to remember new information as the most common first symptom (Alzheimer's-Association, 2016).

A primary feature of AD symptomatology is the progressive decline in cognition (Gelb, 2000). Symptoms of cognitive decline include sporadic episodes of memory loss and impairment in abilities such as concentration and attention, direction, judgement, visuospatial ability (in AD, visuospatial problems suggest that an individual can become disoriented or lost in familiar environments) and language (Vestal et al., 2006). Language impairment or aphasia (the inability to produce or understand speech), is a common issue experienced during the course of AD, as it is passed through from the moderate stage to the severe stage (McKhann et al., 2011).

In 40 to 90% of cases (Hollingworth et al., 2012), AD patients struggle with neuropsychiatric symptoms (NPS), such as depression, anxiety, agitation and delusions, which are significantly more common than in the general population (Steinberg et al., 2008; Steinberg

et al., 2014). Patients with AD are more likely to have NPS which makes it difficult for their

caretakers, and worsens their quality of life (Craig et al., 2005; Steinberg et al., 2014). 2.1.3 Risk factors for Alzheimer’s disease

AD, as previously stated, is a multifactorial disease and develop as a result of not only one cause, but multiple factors (Alzheimer's-Association, 2016).

Age

The main risk factor for AD is aging, but developing AD is not a definite part of aging. AD is most commonly diagnosed in persons over the age of 65 (Alzheimer's-Association, 2016).

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14

Family History

The first-degree relatives (which include parents, siblings or children) of a patient suffering from late-onset AD are at increased risk of developing dementia, with the risk increasing if more than one family member have the disease (Mayeux et al., 1991; Reitz & Mayeux, 2014).

Genetics

Apolipoprotein E (APOE) is a protein that is lipid bound and responsible for the transport of cholesterol into the bloodstream. The APOE gene is localised to the 19q13 chromosome that has been linked to genetic AD in some families with late-onset AD (Struhl & Adachi, 2000; Selkoe, 2001; Reitz & Mayeux, 2014; Alzheimer's-Association, 2016). APOE consists of three isoforms, APOEɛ2, - ɛ3 and - ɛ4 and every individual inherits one isoform from each parent (Raber et al., 2004; Alzheimer's-Association, 2016). The APOEɛ4 isoform is associated with late-onset AD (Selkoe, 2001). The presence of a single APOEɛ4 isoform, increases the risk of developing AD 2.3-fold, whereas there is a 5-fold risk increase in individuals who inherit two copies of this genotype (Kuusisto et al., 1994; Reitz & Mayeux, 2014; Alzheimer’s-Association, 2016). Although the presence of the APOEɛ4 isoform is one of the 3 most common risk factors for AD (Alzheimer's-Association, 2016). The presence of the APOEɛ4 in an individual increases the risk for disease development and early onset of symptoms, not all individuals with the isoform will develop late-onset AD (Balin & Hudson, 2014).

Other deterministic gene mutations associated with AD are amyloidprecursor protein (APP), presenilin 1 (PSEN-1), presenilin 2 (PSEN-2) (Bertram & Tanzi, 2005) and genetic variations of the sortilin-related receptor gene (SORL1), which has been identified as a susceptible gene for late-onset AD (Bettens et al., 2008). Mutations in the APP, PSEN-1 and PSEN-2 genes are also implicated in the pathology of early-onset AD (Reitz & Mayeux, 2014). AD caused by these gene mutations are called “familial Alzheimer’s disease”, where the symptoms may be visible before the age of 60 years. It is possible that the onset of AD symptoms can occur as early as 30 years of age. These deterministic genes will directly cause AD, however, familial AD represents less than 5% of AD cases (Alzheimer's-Association, 2016).

Gender

Based on a study done by Seshadri et al. (2006) in the United States, woman are more likely to develop AD than men.

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15

Mild cognitive impairment

Mild cognitive impairment (MCI) is a syndrome that is defined by cognitive decline. For a patient with MCI, the cognitive decline is greater than that of the normal standard of the expected age and education level, but is not severe enough for daily activities to be interrupted. Normally, for people over the age of 65, the risk of developing MCI is 3 to 19% (Gauthier et al., 2006). Patients with single nucleotide polymorphisms of SORL1 and diagnosed with MCI are at higher risk of developing AD. SORL1 is thus recognised as a potential tool for identifying MCI converting into AD (Piscopo et al., 2015).

Non-genetic, modifiable lifestyle risks

The Alzheimer's-Association (2016) states that the health of the brain is directly linked to a well balanced cardiovascular system. The following are lifestyle risks that can lead to AD:

 Cerebrovascular diseases: The direct underlying mechanism which leads to an increase risk of dementia development through hemorrhagic infarcts, small and large ischemic cortical infarcts, vasculopathies and white matter changes remains unclear. It can be said that white matter hyperintensities and infarcts can cause direct damage to the brain regions responsible for memory function (Reitz & Mayeux, 2014).

 Blood pressure: Studies have shown that hypertension (elevated blood levels) between the ages of 40 – 60 years have been associated with an increase in risk for AD, dementia and cognitive impairment in later stages of life (Swan et al., 1998; Launer et al., 2000; Kivipelto et al., 2001; Whitmer et al., 2005; Reitz & Mayeux, 2014). This is due to the effect that hypertension has on the blood-brain barrier (BBB) where it results in protein extravasation (forcing the protein from the vessel) into the brain tissue (Kalaria, 2010; Reitz & Mayeux, 2014). The presence of protein in the brain tissue can, as a result, cause cognitive impairment through brain cell damage, the reduction in neuronal or synaptic function, apotosis and an increase in Aβ accumulation (Deane et al., 2004; Reitz & Mayeux, 2014).

 Type 2 diabetes: It has been found that type two diabetes may double the risk of late-onset AD development, with the mechanism still unclear (Leibson et al., 1997; Luchsinger et al., 2001; Luchsinger, 2008; Reitz & Mayeux, 2014).

 Body weight: Obesity during midlife is associated with the risk of AD development (Reitz & Mayeux, 2014; Alzheimer's-Association, 2016).

 Smoking: Smoking is a risk factor for AD development, but the exact mechanism by which smoking can cause AD is still unclear. It has been suggested that smoking causes an increase in free radical generation, leading to high oxidative stress and

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16 oxidative damage. Smoking can also lead to cerebrovascular diseases which, as previously stated, enhance the risk of AD (Traber et al., 2000; Reitz & Mayeux, 2014). On the other hand, smoking may have a protective effect in AD where the nicotine of smoking induce the increase of nicotinic acetylcholine receptors (nAChR), which is lost in AD pathology leading to cholinergic deficits (Reitz & Mayeux, 2014). Other risk factors include a history of a traumatic brain injury and low education levels (Reitz & Mayeux, 2014; Alzheimer's-Association, 2016).

2.1.4 Brain structures and pathology in Alzheimer’s disease

In the characteristic pathology of AD, the following brain regions are most affected: the neocortex and hippocampus. These two brain regions are associated with higher mental functions (Francis et al., 1999). The destruction of the underlying chemical pathways that lead to dementia symptoms were first described in the mid 1970’s, when it was observed that the neurons responsible for Ach synthesis and release underwent degeneration that was uneven and severe. The observation was made after, firstly, the discovery of a decrease in levels and activity of certain enzymes (e.g. choline acetyltransferase (ChAT) and AChE) in the limbic as well as the cerebral cortices. Secondly, AD is also associated with a loss in cholinergic cell bodies in the subcortical nuclei which projects to the spetal nuclei and basal forebrain cholinergic system regions (Selkoe, 2001). The disease is thus characterised by synaptic loss, a cholinergichypofunction and degeneration of cholinergic neurons (Blennow et al., 2006; Fisher, 2012).

The presence of deficits in other neurotransmitter systems was identified in the late 1970s and early 1980s. It was realised that AD is highly heterogeneous and does not involve the degeneration of only a single transmitter class of neurons (Selkoe, 2001), but affects multiple regions of the brain (Zubenko et al., 1991).

The pathological characterisation of AD is further hallmarked by the presence of two abnormal protein aggregates (Hyman et al., 2012). Firstly, Aβ deposits (Johnson et al., 2007), which are extracellular (Braak & Braak, 1991) and accumulate outside neurons (Alzheimer’s-Association, 2016), and secondly, aggregated tau protein. These deposits are abnormally hyperphosphorylated tau protein, known as neurofibrillary tangles (NFT), which cause intracellular changes. These NFTs have a specific order of appearance in different brain areas. Their first appearance is in the brainstem and transentorhinal area from where they move to the hippocampus in order to reach the paralimbic and adjacent medial-basal temporal cortex. In the next stage they are found in the cortical association areas before finally reaching the visual and primary sensory-motor areas (Braak & Braak, 1991, Jack &

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17 Holtzman, 2013). The distribution pattern of these NFT’s are pathological features of neurodegeneration in AD (Braak & Braak, 1994; Jack & Holtzman, 2013). The progressive accumulation of these proteins outside (Aβ plaques) and inside (tau tangles) the neurons cause changes that can eventually damage and even cause the death of neurons (Alzheimer's-Association, 2016).

AD’s neuropathologic changes are progressive with the degree of progression correlating with the severity of the clinical disease (Geldmacher, 2007).

2.1.5 Aetiology and pathogenesis of Alzheimer’s disease

In a review by Reitz (2014) it is stated that AD is a complex disease with multifactorial causes and changes. Balin and Hudson (2014) describe AD as a disease with a number of different causal inputs or starting points which result in a definite endpoint: cognitive decline. At the time of death there are several changes present in the brain that is indicative of the disease. These manifestations include the deposits of extracellular Aβ protein in diffuse and neurotic plaques (neurotic plaques contain elements that degenerate neurons). Neurofibrillary tangles are found as extracellular changes where abnormal hyperphosphorylated tau protein and microtubule assembly protein are deposited. There is also widespread neuronal loss and the activation of microglia in the brain (Reitz, 2014). The cause of these manifestations may involve inter alia environmental toxins and infectious agents, which interact in an unknown manner with specific gene aspects of at-risk individuals. For example, an interaction with the APOE gene (previously discussed), may result in neuronal loss, synaptic dysfunction and other characteristics that are experienced during the course of AD (Balin & Hudson, 2014).

Inflammation is considered as an important causative factor for late-onset AD and is associated with neurodegenerative effects (McGeer & McGeer, 2013; Sastra et al., 2011; Balin & Hudson, 2014). Aβ depositions result in the activation of astrocytes and microglia and consequently, in neuroinflammation (Lue et al., 1996, Balin & Hudson, 2014). Microglia are the cells in the brain that are responsible for inflammation and during late-onset AD these cells are activated and often found around amyloid plaques. Activation of microglia cells result in the production of cytokines and reactive oxygen species (Wood, 1998, Balin & Hudson, 2014). Studies have shown that the regular use of non-steroidal anti-inflammatory drugs in the older population may delay the onset of AD; however once cognitive decline has manifested, the use of non-steroidal anti-inflammatory drugs is unsuccessful (Breitner, 1996; Pasinetti, 2002, Balin & Hudson, 2014).

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18 Historically, there are two main hypotheses regarding the aetiology and pathogenesis of AD. The one suggests that the progression of AD is largely influenced by the decrease in ACh production in the synaptic junction (cholinergic hypothesis) (Bartus 2000; Ringman &Cummings, 2006; reviewed in Fisher 2012); while the other suggests that the aggregation of toxic Aβ in the brain contributes to the progression of AD (amyloid hypothesis) (Hardy & Selkoe, 2002). The amyloid hypothesis proposes that treatments that prevent amyloid plaque formation will slow the progress of AD, while the cholinergic hypothesis proposes that treatments that activate the cholinergic system will improve the cholinergic deficits of AD (Stoddard et al., 2014).

2.1.5.1 Amyloid hypothesis

The characteristic neuropathology of AD results from the generation and deposition of Aβ, which arise from a series of cascading events in certain brain areas of an affected individual (Schellenberg, 1995; Claeysen et al., 2012; Balin & Hudson, 2014).

The amyloid plaque is derived from APP, and this plaque consists of deposits of Aβ-peptide, which consists of 39-42 amino acids (Hunt & Turner, 2009, Lichtenthaler, 2011). These peptides are the major protein deposits that are present in AD patients and adult Down syndrome individuals (Masters et al., 1985; Chauhan & Chauhan, 2006). Mutations within the APP are located near and around the APP site which usually promote the proteolytic cleavage of APP by β- and γ- secretase, promoting the generation of Aβ deposits (Citron et

al., 1993; Cai et al., 1993; Hardy & Selkoe, 2002). Mutations on the PS1 and PS2 proteins

increase the processing of APP which also results in the formation of amyloidogenic Aβ (Scheuner et al., 1996; Hardy & Selkoe, 2002).

Aβ can exist as soluble proteins in the cerebrospinal fluid and serum where it is considered a normal metabolic product in both AD patients and healthy individuals (Seubert et al., 1992; Vigo-Pelfrey et al., 1993; Chauhan & Chauhan, 2006). It can also exist as a fibrillar with the ability to aggregate into insoluble fibrils. These fibrils are deposited extracellularly in AD patients’ brains (Chauhan & Chauhan, 2006). Aβ-oligomers cause neurotoxicity and neurodegeneration which is responsible for the behavioural and functional shortages present in AD(Hardy & Selkoe, 2002).

The series of pathogenic events that leads to AD as described by the amyloid hypothesis begin with missense mutations in the APP, PS1 or PS2 genes which lead to an increase of Aβ production and accumulation. Oligomerisation of Aβ peptides takes place as well as deposition of Aβ as diffuse plaques. Generated Aβ oligomers have effects on synapses which activate microglial and astrocytic cells resulting in neuroinflammation (as previously

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19 described). Neuroinflammation causes progressive synaptic and neurotic injury, however the Aβ oligomers not only activate the microglia and astrocytes, but it can cause direct injury in the brain through damage to the synapses and neuritis. Injured synapses and neuritis alter kinase and phosphatase activities, producing tangles. These tangles cause widespread neuronal and neurotic dysfunction and cell death, leading to dementia (Hardy & Selkoe, 2002).

2.1.5.2 Cholinergic hypothesis

Working memory, conscious awareness and attention is dependent on the functioning of well balanced cholinergic pathways in the basal and rostral forebrain (Perry et al., 1999; Terry & Buccafusco, 2003). In AD there is an abnormality in these cholinergic pathways which correlates with the level of cognitive decline (Bartus, 2000; Terry & Buccafusco, 2003). In the cholinergic hypothesis, the following is stated: During the course of AD, a loss of cholinergic neurotransmission in the cerebral cortex and other areas occurs as a result of severe damage to the cholinergic neurons in the basal forebrain (this can be detected both histopathologically, by a loss of neurons, and neurochemically by a loss of marker enzymes for ACh synthesis and degradation). The characteristic symptoms of AD, such as memory loss, cognitive and non-cognitive symptoms, are the result of these cerebral cholinergic deficits (Frölich, 2002).

It has been shown that in AD, there is a substantial loss of the enzyme ChAT which is responsible for the synthesis of ACh (Bowen et al., 1976, Davies & Maloney, 1976; Perry et

al., 1977; Francis et al., 1999) with a reduction in ACh release (Nilsson et al., 1986; Francis et al., 1999) and choline uptake (Rylett et al., 1983; Francis et al., 1999) as well as the loss

in cholinergic perikarya from the nucleus basalis of Meynert, resulting in cholinergic deficits (Whitehouse et al., 1982, Francis et al., 1999). ACh is a neurotransmitter that binds to nicotinic (nAChRs) and muscarinic Ach receptors (mAChRs). Both nicotinic and muscarinic receptors are involved in cognitive processes and in AD, both nAChRs and mAChRs are affected (Blennow et al., 2006; Giacobini & Becker, 2007; Fisher, 2012). For example, a reduced expression of nAChRs occurs in AD (Maelicke et al., 2001).

There are critics of the cholinergic hypothesis which suggest that previous studies focused only on end stage AD while the characteristics of MCI or mild AD were not investigated (Terry & Buccafusco, 2003). For example, a study done by Davis et al. (1999) reported that the activity of AChE and ChAT was not reduced in the neocortical tissue of post mortem mild AD patients that was recently diagnosed. Furthermore, a study done by DeKosky et al. (2002) on MCI and mild AD patients have failed to identify any reduction in ChAT activity.

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20 The definite aetiology of AD is still uncertain which makes it difficult to find effective means of delaying or halting disease progression (Fisher, 2012).

2.1.5.3 Oxidative stress

Oxygen (O2) is essential for normal living. It has a high redox potential, allowing it to easily accept electrons from substrates (Gandhi & Abramov, 2012). In a functioning system, electrons that are released from the respiratory chain react with O2 to produce an oxygen-based free radical anion, better known as superoxide (O2•-). Superoxide dismutase (SOD) converts the O2•- to hydrogen peroxide (H2O2). H2O2 is a potent oxidising agent, reacting with metals and ferrous iron (Fe2+) to produce hydroxal radicals (OH•), which are highly reactive. O2•-also cross-reacts with nitric oxide (NO) for the production of NO2• (Chauhan & Chauhan, 2006). O2•-, OH• and H2O2 are examples of different reactive oxygen species (ROS). These species are generally inactivated through glutathione peroxidase which uses glutathione (GSH) as its cofactor. A GSH deficiency can lead to a decrease in the brain’s ability to clear ROS, causing oxidative stress and cell death (Chance et al., 1979; Youdim et al., 2006). This is because these unstable radicals (that are constantly in search of electrons) are toxic, reacting with proteins, nucleic acid and lipids, resulting in abnormal cellular function. Oxidative damage to cellular components causes alterations in membrane functions with excessive damage leading to cell death (Bandyopadhyay et al., 1999).

Oxidative stress is thus a state that is induced by the dysfunction of the antioxidant system or the generation of an excess ROS (Andreyev et al., 2005). The neuronal degeneration in AD pathology is greatly affected by oxidative stress. Contributing factors that increase oxidative stress in AD include soluble Aβ, Aβ fibrils, mitochondrial abnormalities, aging and NFT. Another contributing factor involved in AD’s neuronal degeneration is the imbalance in oxidative homeostasis, as it leads to an increase in lipid peroxidation (Chauhan & Chauhan, 2006). Aging is associated with an increase in ROS production (Chauhan & Chauhan, 2006; Zhu et al., 2007), providing at least in part, some explanation of why aging is such an important risk factor for developing AD.

Metals as contributors to oxidative stress

Metals catalyse redox reactions, inducing oxidative stress (Birben et al., 2012); on the other hand, antioxidant defences consist of the storing and transportation of metals, such as iron, in forms that would not catalyse the formation of reactive radicals. Therefore, with an increase in iron availability, the rate of free radical reactions can accelerate (Valko et al., 2006).

Design and synthesis of dual acting carbamate inhibitors of acetylcholinesterase and monoamine oxidase